It is well known that certain proteinaceous compounds have the ability to substitute in a highly effective manner for sugar in giving foods and beverages a sweet taste. The simplest of these examples is aspartame, which is a dipeptide derivative and currently on the market. However, two much more complex proteins, monellin and thaumatin have been isolated from plant sources.
Thaumatin is isolated from Thaumatococcus daniellii, a West African plant having triangular shaped fruit at ground level. The natural protein product, thaumatin, has an average sweetness of 2500 times that of sucrose and has been marketed under the trademark Talin. The three-dimensional structure of this protein has been studied and the results published by De Vos, A. M., et al., Proc Natl Acad Sci USA (1985) 82:1406-1409. At least five highly related forms of thaumatin (I, II, III, b and c) have been identified (Higginbotham, J. D. in Developments in Sweeteners-1 (Hough, C.A.M. et al., eds.) Applied Science Publications, London 1979, pp. 87-123. Furthermore, the gene encoding the Thaumatin II protein has been cloned and its sequence has been determined by Edens, L., et al., Gene (1982) 18:1. Thaumatin has also been produced recombinantly in bacteria (Edens, L., et al., Gene (1982) 18:1) and yeast (Edens, L., et al., Cell (1984) 37:629); Lee, et al, Biochemistry (1988) 27:5101).
The other protein is isolated from "Serendipity Berries" of the West African Plant Dioscoreophyllum comminisii. The amino acid sequence of monellin is known, and the three-dimensional structure of this protein has been determined by Ogata, C., et al., Nature (1987) 328:739-742. Monellin has been characterized by Morris et al., J Biol Chem (1973) 248:534-539, and by others; Cagan, Science (1973) 181:32-35; Bohak and Li, Biochim Biophys Acta (1976) 427:153-170; Hudson and Beeman, Biochem Biophys Res Comm (1976) 71:212-220; Van der Wel and Loeve, FEBS Lett (1973) 29:181-183; Frank and Zuber Hoppe-Seyler's Z Physiol Chem (1976) 357:585-592; Morris and Cagan, Biochim Biophys Acta (1972) 261:114-122. U.S. Pat. No. 3,998,798 describes the preparation of natural monellin.
The known amino acid sequence of the A and B chains of natural monellin is shown in FIG. 1. It is a two chain protein, one "A" chain containing 45, and the other "B" chain, 50 amino acid residues. The three-dimensional conformation of the protein, shown in FIG. 2, is evidently essential for its activity because when native monellin is heated to 90.degree. C. at neutral pH or to 50.degree. C. at acidic pH and then cooled, the sweetness is destroyed. The B chain containing 50 amino acids is intimately associated with the A chain of 45 amino acids in such a way that there are many interchain interactions. Heating of the protein, evidently dissociates the chains in such a way that they are incapable of reforming into the appropriate conformation.
The parent applications herein, now published as PCT application WO 88/10265 and European application EP 318,580 and EP 323,489, and Kim, S.-H., et al., Protein Engineering (1989) 2:571-575 describe the apparent stabilization of the conformation by synthesizing appropriate portions of the A and B chains as part of a single protein, and the recombinant production of the single chain form of monellin. Various amino acid sequences can be used to form a linkage between amino acid 46 (Ile) of the B chain with amino acid 6 (Gly) of the A chain. As described in these references, limited modifications of the A and B chain portions of the resulting single chain monellin may also be included. These modifications are made possible by the availability of the gene encoding the single chain sequence, which was synthesized using commercially available methods.
Monellin and thaumatin are the only proteins currently known which have a high specificity for human sweet taste receptors, thus resulting in a highly sweetening effect. Comparison of thaumatin and monellin amino acid sequences shows no significant homology either in amino acid sequence or in their three dimensional backbone structures (Kim S.-H. et al. Trends in Biochemical Sciences (1988) 13:13). However, it may be that other as yet undiscovered proteins with this property exist and it is quite likely that only a limited region of the three dimensional structure is required for the human taste receptor specificity displayed by these proteins.
It has now been found that in addition to recombinant manufacture of substantial quantities of monellin and of thaumatin using recombinant methods, edible parts of plants can be provided in "naturally" sweetened form by producing these edible materials as fruits, seeds or vegetative parts of transgenic plants wherein the transgenic plants are modified from their natural counterparts by introduction of an expression system for the thaumatin or monellin gene or the single chain monellin gene. The choice of control sequences in the expression system also permits targeting of the expression to the desired plant tissue, and expression at the appropriate time in development.